Coding and traceability for cells, tissues and organs for transplantation.

Modern transplantation of cells, tissues and organs has been practiced within the last century achieving both life saving and enhancing results. Associated risks have been recognized including infectious disease transmission, malignancy, immune mediated disease and graft failure. This has resulted in establishment of government regulation, professional standard setting and establishment of vigilance and surveillance systems for early detection and prevention and to improve patient safety. The increased transportation of grafts across national boundaries has made traceability difficult and sometimes impossible. Experience during the first Gulf War with mis-identification of blood units coming from multiple countries without standardized coding and labeling has led international organizations to develop standardized nomenclature and coding for blood. Following this example, cell therapy and tissue transplant practitioners have also moved to standardization of coding systems. Establishment of an international coding system has progressed rapidly and implementation for blood has demonstrated multiple advantages. WHO has held two global consultations on human cells and tissues for transplantation, which recognized the global circulation of cells and tissues and growing commercialization and the need for means of coding to identify tissues and cells used in transplantation, are essential for full traceability. There is currently a wide diversity in the identification and coding of tissue and cell products. For tissues, with a few exceptions, product terminology has not been standardized even at the national level. Progress has been made in blood and cell therapies with a slow and steady trend towards implementation of the international code ISBT 128. Across all fields, there are now 3,700 licensed facilities in 66 countries. Efforts are necessary to encourage the introduction of a standardized international coding system for donation identification numbers, such as ISBT 128, for all donated biologic products.

In vitro culture and differentiation of osteoblasts from human umbilical cord blood.

It is well accepted that human umbilical cord blood (UCB) is a source of mesenchymal stem cells (MSCs) which are able to differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. The aim of this study was to isolate MSCs from human UCB to determine their osteogenic potential by using different kinds of osteogenic medium. Eventually, only those MSCs cultured in osteogenic media enriched with vitamin D(2) and FGF9, were positive for osteocalcin by RT-PCR. All these cells were positive for alizarin red, alkaline phosphatase and Von Kossa. The results obtained from RT-PCR have confirmed that osteogenesis is complete by expression of the osteocalcin marker. In conclusion, vitamin D(2), at least in vitro, may replace vitamin D(3) as an osteogenic stimulator factor for MSC differentiation.

Performance of an algorithm for the reentry of volunteer blood donors deferred due to false-positive test results for antibody to hepatitis B core antigen.

BACKGROUND: Blood donor testing for antibody to hepatitis B core antigen (anti-HBc) has been used in the United States for more than 20 years as a surrogate to prevent transmission by transfusion of non-A, non-B hepatitis, as a human immunodeficiency virus surrogate, and to reduce transmission of hepatitis B virus (HBV). Nonspecific anti-HBc assays have caused deferral of hundreds of thousands of otherwise qualified donors. A more specific anti-HBc test and a sensitive HBV DNA test should permit donor reentry after false-positive anti-HBc. STUDY DESIGN AND METHODS: A total of 1324 otherwise eligible volunteer donors, deferred for anti-HBc reactivity on more than one occasion, were recruited from four collection facilities. They were tested using a licensed, more specific anti-HBc test, a licensed hepatitis B surface antigen (HBsAg) test, and a licensed HBV DNA assay with a 95 percent limit of detection of not more than 10 copies per mL. RESULTS: From 11 to 32 percent of donors contacted by participating sites entered the study. Overall, 488 (37%) of the donors were negative on the more specific anti-HBc test. The proportion of putative false-positive samples varied according to the test responsible for the original deferral. A single donor, negative for the presence of anti-HBc and HBsAg, was positive for the presence of HBV DNA in one of three replicates. Repeat testing of this donor 10 months later was negative for the presence of all markers of HBV infection, and the donor had a history of HBV vaccination with documented postimmunization anti-HBs seroconversion 10 years before her anti-HBc deferral, and was considered HBV DNA false positive. CONCLUSION: These data support reentry of donors with false-positive anti-HBc results on the relatively nonspecific assays that have been in use in the United States for more than 20 years.

Donor derived malignancy following transplantation: a review.

Organ and tissue transplant is now the treatment of choice for many end stage diseases. In the recent years, there has been an increasing demand for organs but not a similar increase in the supply leading to a severe shortage of organs for transplant resulted in increasing wait times for recipients. This has resulted in expanded donor criteria to include older donors and donors with mild disease. In spite of implementation of more stringent criteria for donor selection, there continues to be some risk of donor derived malignancy. Malignancy after transplantation can occur in three different ways: (a) de-novo occurrence, (b) recurrence of malignancy, and (c) donor-related malignancy. Donor related malignancy can be either due to direct transmission of tumor or due to tumor arising in cells of donor origin. We will review donor related malignancies following solid organ transplantation and hematopoeitic progenitor cell transplantation. Further, we will briefly review the methods for detection and management of these donor related malignancies.

Human tissue oversight in hospitals: an AABB survey.

BACKGROUND: Reports of human tissue allograft-transmitted infections have underscored the need for better accounting of allografts in health-care facilities. The Joint Commission on Accreditation of Healthcare Organizations (JCAHO) implemented new storage and issuance tissue standards for hospital oversight as of July 1, 2005. This study sought to survey hospital tissue responsibilities. STUDY DESIGN AND METHODS: The AABB Tissue Task Force conducted a Web-based survey distributed to all 904 hospital institutional members in January 2005. The survey asked about tissue type used, breadth of responsibility, hospital department involvement, and views on AABB involvement. Data from 402 of 904 (45%) respondents were tabulated and analyzed. RESULTS: Among the 402 respondents, 325 (81%) used allogeneic and/or autologous human tissue. The most frequently used tissues were musculoskeletal (n = 240, 74%) and skin (n = 169, 52%) allografts. The department of surgery (e.g., operating room; n = 245, 76%) most often had responsibility for tissue use, followed by the blood bank (i.e., transfusion service; n = 164, 51%); surgery most frequently had responsibility for all tissue types except peripheral blood progenitor cells. Only 32 of 402 (8%) respondents had plans for increased oversight in the next 12 months; 129 of 178 (72%) thought there was a role for AABB in developing guidance on hospital tissue responsibilities. CONCLUSIONS: In this survey, most AABB member hospital respondents indicated facility use of allogeneic and/or autologous tissues. Although tissue allograft responsibility by surgery was extensive, hospital blood banks also had significant involvement. Few blood banks, however, plan increased oversight in the near future. Given JCAHO standards, blood banks have an opportunity to assist their hospital in planning for assigned tissue responsibilities and oversight to ensure patient safety.

Improve islet yields and quality when clinical grade pancreata are preserved by the two-layer method.

BACKGROUND: Research grade pancreata preserved by the two-layer method (TLM) yield significantly greater numbers of islets than organs stored with University of Wisconsin solution (UW). The goal of this study was to determine whether this would hold true for pancreata that meet selection criteria for clinical grade organs. METHODS: Pancreata were chosen based upon a pre-defined set of criteria used for selecting clinical grade pancreata. Thirteen of these organs were preserved in UW and five pancreata were preserved by the TLM. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: The average preservation time was significantly longer for organ preserved with TLM (9.5 + 2.0 h) as compared to UW (5.8 + 0.6 h, p = 0.015). The pancreata of TLM group resulted in a significant increase in islet yields (3588 +/- 500 vs. 2536 +/- 312 IE/g pancreas, p<0.05). Visual scoring of islets indicated that islets were better from TLM group (8.3 +/- 0.3 vs. 7.3 +/- 0.2), and islet survival rates after culture were higher from organs stored with the TLM (87 +/- 17 vs. 55 +/- 7.4, p<0.05). Other parameters such as viability, insulin content, and stimulation index were similar between the two groups. All the preparations from the TLM group, but only 54% of preparations from the UW group, qualified for islet transplantation. The two recipients receiving islets from TLM group, daily insulin requirements were reduced and C-peptide levels were increased. CONCLUSION: Compared to storage with UW, exposure of pancreata to the TLM resulted in greater islet yields and improved quality of islets despite longer preservation period. Consequently, pancreata that meet clinical grade status should be preserved by the TLM prior to islet isolation.

Preventing disease transmission by deceased tissue donors by testing blood for viral nucleic acid.

Nucleic acid testing (NAT) has reduced the risk of transmitting infectious disease through blood transfusion. Currently NAT for HIV-1 and HCV are FDA licensed and performed by nearly all blood collection facilities, but HBV NAT is performed under an investigational study protocol. Residual risk estimates indicate that NAT could potentially reduce disease transmission through transplanted tissue. However, tissue donor samples obtained post-mortem have the potential to produce an invalid NAT result due to inhibition of amplification reactions by hemolysis and other factors. The studies reported here summarize the development of protocols to allow NAT of deceased donor samples with reduced rates of invalid results. Using these protocols, inventories from two tissue centers were tested with greater than 99% of samples producing a valid test result.

Probability of viremia with HBV, HCV, HIV, and HTLV among tissue donors in the United States.

BACKGROUND: Tissue-banking organizations in the United States have introduced various review and testing procedures to reduce the risk of the transmission of viral infections from tissue grafts. We estimated the current probability of undetected viremia with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and human T-lymphotropic virus (HTLV) among tissue donors. METHODS: Rates of prevalence of hepatitis B surface antigen (HBsAg) and antibodies against HIV (anti-HIV), HCV (anti-HCV), and HTLV (anti-HTLV) were determined among 11,391 donors to five tissue banks in the United States. The data were compared with those of first-time blood donors in order to generate estimated incidence rates among tissue donors. The probability of viremia undetected by screening at the time of tissue donation was estimated on the basis of the incidence estimates and the window periods for these infections. RESULTS: The prevalence of confirmed positive tests among tissue donors was 0.093 percent for anti-HIV, 0.229 percent for HBsAg, 1.091 percent for anti-HCV, and 0.068 percent for anti-HTLV. The incidence rates were estimated to be 30.118, 18.325, 12.380, and 5.586 per 100,000 person-years, respectively. The estimated probability of viremia at the time of donation was 1 in 55,000, 1 in 34,000, 1 in 42,000, and 1 in 128,000, respectively. CONCLUSIONS: The prevalence rates of HBV, HCV, HIV, and HTLV infections are lower among tissue donors than in the general population. However, the estimated probability of undetected viremia at the time of tissue donation is higher among tissue donors than among first-time blood donors. The addition of nucleic acid-amplification testing to the screening of tissue donors should reduce the risk of these infections among recipients of donated tissues.

Polyphenol, an extract of green tea, increases culture recovery rates of isolated islets from nonhuman primate pancreata and marginal grade human pancreata.

Investigations indicate that an extract of green tea, polyphenol, can significantly increase the culture survival rate of rat islets without deteriorating their functionality. In this study, we examined the effect of adding polyphenol to islets isolated from human pancreata and nonhuman primate pancreata. Islets were isolated from human pancreata that did not meet criteria for clinical transplantation (n = 6) and from nonhuman primate pancreata (n = 5). The islets were cultured in CMRL-1066 + 10% FCS with the addition of 0, 30, 60, 125, 250, or 500 microg/ml of polyphenol. After 24 or 48 h of culture, islet yield, viability, purity, morphology, and stimulation index was assessed. RT-PCR and Western blot analysis were also performed to assess the expression levels of the apoptotic related genes, Bcl-2 and BAX. After 24 h of culture, islet yields were significantly higher in cultures supplemented with 30-250 microg/ml of polyphenol than in cultures without polyphenol. After 48 h of culture, significant differences in islet numbers were observed with polyphenol concentrations of 125 microg/ml (p < 0.01) and 250 microg/ml (p < 0.01). However, no significant differences were noted in islet viability, purity, morphology, and stimulation index at each time point with or without polyphenol. RT-PCR and Western blot analysis of the islets indicated that Bcl-2 levels increased by 2.5-fold and BAX levels decreased by twofold in cultures supplemented with polyphenol. This resulted in BAX/Bcl-2 ratios that were lower in polyphenol-supplemented cultures than with control cultures. Polyphenol increases culture recovery rates by precluding islet apoptosis.

Cord blood units collected at a remote site: a collaborative endeavor to collect umbilical cord blood through the Hawaii Cord Blood Bank and store the units at the Puget Sound Blood Center.

BACKGROUND: Umbilical cord blood is a useful source of hematopoietic stem cells, especially because compared to equivalent HLA-matched stem cells from unrelated adult donors. A network of community collection sites targeted at particular ethnic groups and serviced by a central processing and storage facility can maximize the genetic diversity of banked cord blood units (CBUs) in a cost-effective fashion. STUDY DESIGN AND METHODS: The present study compared CBUs collected near the Puget Sound Blood Center in Seattle, WA, with those collected in Honolulu, HI, and processed in Seattle. Evaluated variables include collection volume, total nucleated cell count, cellular viability, CD34+ cell count, clonogenic activity, and donor race for a total of 1646 CBUs received from July 1998 through November 2002. RESULTS: CBUs from the two sites did not differ with regard to volume or total nucleated cells. Those from Hawaii had significantly longer transit times (p < 0.001) and lower whole cord blood cell viability. However, the numbers of CFU and viable CD34+ cells were not affected by remote collection. CBUs screened from Seattle were largely from Caucasian donors, whereas over 85 percent of those from Honolulu were from donors of Asian-Pacific Islander or mixed ethnicity. CONCLUSION: These studies demonstrate the feasibility of long-distance umbilical cord blood banking. Arrangements such as those described here could be used to help target cost-effective collection from minority populations and increase the HLA and ethnic diversity for CBUs.

Donor age and gender are the strongest predictors of marrow recovery from cadaveric vertebral bodies.

The purpose of this retrospective analysis was to determine whether there were donor factors that were useful for predicting the yield of nucleated cells from marrow derived from cadaveric vertebral bodies. An analysis of 132 donors over a 6-year period was performed. The average number of vertebral bodies procured from each donor was 10.2 +/- 1.6 (range 5-14). The total number of nucleated cells recovered per donor ranged from 24 x 10(9) to 160 x 10(9) with an average recovery of 69 +/- 28 x 10(9) cells. The cell viability of the recovered cells was > 95%. The average age of the donors was 33 +/- 14 years (mean +/- SD; range 12-65) with an average weight of 169 +/- 41 lb (range 82-308 lb). Males comprised 68% of the donor population. The average number of days from admission to death was 1.9 +/- 1.7 with a range of 1-11.4 days and the interval between asystole and procurement averaged 3.1 +/- 2.3 h (range (0.1-14.7 h). The majority of donors died from head trauma due to an intracranial bleed, gunshot wound, or closed head injury. Regression analysis of the data indicated that the total nucleated cell yield tended to decrease with increasing time between hospital admission and death. The data also indicated that in general female donors yielded lower cell numbers independent of age and male donors under 30 years of age yielded the highest number of cells.

Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation, as assessed by the Edmonton Isolation Protocol.

BACKGROUND: Current techniques for isolating islets require that pancreata stored with University of Wisconsin solution (UW) are processed within 12 hours of cold storage. In this study, we hypothesized that the two-layer method (TLM) could extend the acceptable preservation period of pancreata before islet isolation and increase islet yields. METHODS: In the first experimental set, eight pancreata were maintained for an average of 8.3+/-1.2 hours in UW and transferred into the TLM for an additional 14.3+/-1.1 hours for a total cold ischemic period of 22.6+/-1.6 hours (prolonged TLM). Four pancreata were maintained as a control group in UW alone for a total of 21.3+/-2.0 hours. In the second experimental set, six pancreata were maintained for an average of 6.4+/-1.8 hours in UW followed by 4.8+/-0.8 hours with the TLM for a total preservation time of 11.3+/-2.5 hours (short TLM). The control organs for the short TLM group were stored for an average of 9.5+/-1.3 hours in UW alone. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: Between each group of the two experimental sets, there was no significant difference in donor-related factors (i.e. gender, age, body mass index [BMI], etc.). The TLM as compared with UW preservation resulted in a significant increase in islet yields postpurification for both short (3,353+/-394 islet equivalents [IE] vs. 2,027+/-415 IE; mean+/-SEM) and prolonged (2,404+/-503 IE vs. 514+/-180 IE) periods of storage. Furthermore, islet yields after prolonged storage with the TLM were not significantly different from organs maintained for only a short period with UW (P=0.17). The quality of islets as assessed by size, postculture viability, survival rates, insulin content, and insulin secretion were similar for each of the four groups. CONCLUSION: In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.

Comparative yield of HCV RNA testing in blood donors screened by 2.0 versus 3.0 antibody assays.

BACKGROUND: Two HCV antibody tests (EIA 2.0 [EIA2], Abbott; and the Version 3.0 ELISA [EIA3], Ortho) are currently licensed for screening of US blood donors. Testing of donors for HCV RNA allows comparison of the sensitivities of the two antibody-screening assays. STUDY DESIGN AND METHODS: All allogeneic blood donations at 13 US test sites were screened for HCV RNA by testing plasma minipools using an investigational assay (COBAS AmpliScreen HCV test, v2.0, Roche Molecular Systems). Some sites screened for HCV antibody by EIA2 and some used EIA3. The frequency of RNA-positive and antibody-negative (RNA-pos and Ab-neg) donations among donors screened by each antibody assay was compared. Antibody appearance was assessed in a donor follow-up study. RESULTS: A total of 5.51 x 10(6) donations were screened for HCV RNA. Of these, 2.27 million were screened for antibody by EIA2, and 3.24 million by EIA3. Twenty-three donations were HCV RNA-pos and Ab-neg. The frequency of RNA-pos and Ab-neg donations was higher among donations screened by EIA2 (1 in 134,000), compared to those screened by EIA3 (1 in 540,000) (p = 0.001). Of the 17 RNA-pos and Ab-neg donations identified by test sites that used EIA2, 14 were retested by EIA3 and 10 (71%) were reactive. Most RNA-pos and Ab-neg donors appear to be in the process of seroconversion. Donors that were initially EIA2-negative and EIA3-reactive showed a more prolonged pattern of seroconversion compared to those that were initially nonreactive by both antibody assays. Four donors were EIA2-negative, EIA3-reactive, and RIBA-indeterminate (c33c) for at least 90 days, 1 for more than 317 days. CONCLUSION: EIA3 would have detected the majority of RNA-positive donations missed by EIA2. Some RNA-positive donors are EIA2-negative and EIA3-reactive for a prolonged period of time.

Banking of osteochondral allografts. Part I. Viability assays adapted for osteochondrol and cartilage studies.

The aim of this study was to adapt a reliable, reproducible and simple viability assay for cartilage and osteochondral studies. The previous assays (radioisotope uptake, assessment of matrix components, histological methods, oxygen consumption etc.) were complex, laborious, time consuming or suffer from difficulty of interpretation. MTT assay was chosen because it has been widely and successfully used in different cell and tissue studies, but has not been published on human solid articular cartilage. Fresh intact cartilage samples of human tali were tested to investigate the assay. The reliability of the MTT assay was also tested by an fluorescent dye combination. The MTT assay is based on the production of purple formazan pigment from methyltetrazolium salt by the mitochondrial enzymes of viable chondrocytes. The enzyme kinetics of the reaction was also investigated because it was unknown in the case of cartilage. The amount of pigment formed can be measured by spectrophotometry after extraction by methyl cellosolve. The color density is proportional to mitochondrial enzyme activity, reflecting the number of viable chondrocytes. The optimal reagent concentration, biopsy size, and incubation period were established. There is a linear relationship between the cartilage weight and the pigment production activity. A 9.8% nonspecific raction was observed in the negative controls. The enzyme kinetics of the reaction was also investigated. The MTT clevage up to 0.1% (w/v) follows the Michaelis kinetics. We calculated the Michaelis constant (2835 +/- 130 microM), the maximal velocity (36 +/- 3.2 x 10(-5)microMsec(-1)) and the velocity constant (1.27 +/- 0.2 x 10(-7)sec(-1)) of the reaction. The latter is a significant marker for each tissue type. The viability of cartilage was also assessed and calculated by a fluorescent dye combination comprising 1 microg/ml propidium iodide (PI) and 4 microM/ml SYTO-16 stains. The PI stains dead cells (red fluorescence), the SYTO-16 stains live cells (green fluorescence). The staining can be visualised simultaneously, and the live/dead ratio can be calculated by image analysis software from saved image files. The MTT assay is a simple, non-expensive, efficient, reliable, reproducible, sensitive viability test for cartilage studies. The MTT reduction assay and the staining method were corrobative.